1. Field of the Invention
The present invention primarily relates to specific uses of a composition containing a roasted extract and xanthohumol for health-promoting purposes.
2. Discussion of Background Information
It is well known that xanthohumol has very diverse positive effects on human and animal health. For example, besides antimicrobial effects, preventive activity against a wide range of diseases by various mechanisms is being observed.
For example, cancer-preventive activity, protective action against osteoporosis, metabolic syndrome, diabetes and cardiovascular diseases have been reported.
It should be mentioned that these sub-divisions cannot be stringent. For example, anti-oxidative effects are just as positive in the prevention of tumors as, for example, with the metabolic syndrome or other diseases that up to now have not been investigated in connection with xanthohumol.
A large number of studies describes cancer-preventing mechanisms and were conducted in vitro, mostly in cell culture. Separate reference is made to the in vivo tests. The genesis of a tumor is a multi-factorial phenomenon; many mechanisms work interactively, many are known, many unknown. A frequently applied classification summarizes the early molecular events as “tumor initiation”, the mechanisms of the development and establishment of a tumor as “tumor promotion” and the growth of a tumor as “tumor progression”. The effect of xanthohumol was examined in a wide diversity of systems in line with the many mechanisms; these have been summarized and described as follows.
Phase I enzymes denote the enzymes that are responsible for the metabolic transformation of pro-carcinogenic substances into reactive carcinogenic metabolites, i.e. toxification. The inhibition of such enzymes by xanthohumol in tumor cells in culture, in rat liver and in recombinant human enzyme has been described by Henderson et al. (2000), Xenobiotica, 30, 235-251; Miranda et al. (2000), Cancer Lett., 149, 21-29; Miranda et al. (2000), Drug Metab Dispos., 28, 1297-1302 and Gerhauser et al. (2002), Mol. Cancer Ther., 1, 959-969.
Phase II enzymes are those which are responsible in the organism for the conjugation of reactive, carcinogenic metabolites, secreting them from the cells towards detoxification. The stimulation of this class of enzyme is hence protective. Stimulation by way of xanthohumol in human liver tumor culture cells has also been described by Henderson et al. (2000), Xenobiotica, 30, 235-251; Miranda et al. (2000), Cancer Lett., 149, 21-29; Miranda et al. (2000), Drug Metab Dispos., 28, 1297-1302 and Gerhauser et al. (2002), Mol. Cancer Ther., 1, 959-969.
Cellular oxidation is a process necessary to the organism to maintain life (securing of energy, respiration, bacterial defense). During its course, oxidative damage is caused that the healthy organism will eliminate using its own repair systems. If these systems are inadequate, whether because of defects, because of an excess of oxidative injury, as for example with chronic inflammations, reactive, oxidative molecules can react with the body's own macromolecules and lead to defects and mutation that may be the cause of a tumor as well as of other diseases. For this reason, anti-oxidative characteristics are highly significant for the prevention of tumors and other cellular injuries. Xanthohumol inhibits the formation and the action of reactive oxygen and hydrogen molecules in cell cultures, in rat liver fractions and in enzymatic model systems, as described by Miranda et al. (2000), J. Agric. Food Chem., 48, 3876-3884; Rodriguez et al. (2001), Food Chem. Toxicol., 39, 437-445; Gerhauser et al. (2002), Mol. Cancer Ther., 1, 959-969, Stevens et al. (2003) Chem. Res. Toxicol., 16, 1277-1286 and Vogel et al. (2008), Natural and non-natural prenylated chalcones: Synthesis, cytotoxicity and anti-oxidative activity. Bioorg. Med. Chem., e-pub. It should be said that xanthohumol is not able to intercept the stable diphenylpicrylhydrazyl (DPPH) radical in direct chemical interaction (Gerhauser et al. (2002), Mol. Cancer Ther., 1, 959-969; Dietz et al. (2005), Chem Res. Taxieal., 18, 1296-1305; Gerhauser and Frank (2005), Mol. Nutr. Food Res., 49, 821823; Jung et al. (2005), Arch. Pharm Res., 28, 534-540). It would appear that the antioxidative effect of xanthohumol is derived from indirect cellular mechanisms.
As already described, inflammatory processes are often the cause of the formation of oxidative noxious substances that may be responsible for a number of diseases. Not only this, substances are detected in many human tumors that indicate the involvement of inflammatory processes. For this reason, anti-inflammatory agents act preventively against tumors. In Gerhauser et al. (2002), Mol. Cancer Ther., 1, 959-969; Zhao et al. (2003), Biol Pharm. Bull., 26, 61-65 and Cho et al. (2008), Int. Immunopharmacol., 8, 567-573, xanthohumol is described in cell culture as being anti-inflammatory; in Monteiro et al. (2008) Xanthohumol inhibits inflammatory factor production and angiogenesis in breast cancer xenografts, J. Cell Biochem, e-pub, the inhibitive effect on inflammation was also established in vivo in mice after the oral administration of a dose of 4-6 mg/kg per day for 60 days in drinking water.
Estrogen is a growth factor and provides degenerate cells with an edge on growth. For this reason, anti-estrogens are considered to have a cancer-preventive action with tumors that depend on hormones. In Gerhauser et al. (2002), Mol. Cancer Ther., 1, 959-969; Effenberger et al. (2005), J. Steroid Biochem. Mol. Biol., 96, 387-399; Monteiro et al. (2006), J. Agric. Food Chem., 54, 2938-2943 and Monteiro et al. (2007) J. Steroid Biochem. Mol. Biol., 105, 124130, the anti-estrogen effect of Xanthohumol in vitro has been described; this is also described in Gerhauser et al. (2002), Mol. Cancer Ther., 1, 959-969 in an organ culture model of the mouse mammary gland. In an in vivo test on rats, it was also possible to detect anti-estrogen action after the oral application of three×100 mg/kg xanthohumol over three days (Gerhauser and Frank (2005), Mol. Nutr. Food Res., 49, 821823).
A significant feature in the genesis and growth of a tumor is the pronounced growth of the tumor cells. Inhibition of proliferation serves to prevent cancer and to provide tumor therapy. For this reason anti-proliferative substances are sought that inhibit the growth of tumor cells, yet at the same time do not destroy healthy cells. In many cases, of course, this is a question of dose and the onset time of substance action. Xanthohumol has been described as inhibiting growth in 24 different tumor cell lines (Miranda et al. (1999), Food Chem. Toxicol., 37, 271-285; Gerhauser et al. (2002), Mol. Cancer Ther., 1, 959-969; Herath et al. (2003), Chem Pharm. Bull. (Tokyo), 51, 1237-1240; Dietz et al. (2005), Chem Res. Taxieal., 18, 1296-1305; Goto et al. (2005), Cancer Lett., 219, 215-222; Lust et al. (2005), Mol. Nutr. Food Res., 49, 844850; Pan et al. (2005), Mol. Nutr. Food Res., 49, 837-843; Albini et al. (2006), FASEB J., 20, 527-529; Oelmulle et al. (2006), Phytomedicine., 13, 732-734; Colgate et al. (2007), Cancer Lett., 246, 201-209; Dell'Eva et al. (2007), Cancer, 110, 2007-2011; Lee et al. (2007), Arch Pharrn. Res., 30, 14351439; Monteiro et al. (2007), J. Steroid Biochem. Mol. Biol., 105, 124130; Delmulle et al. (2008), Phytother Res., 22, 197-203; Monteiro et al. (2008), Xanthohumol inhibits inflammatory factor production and angiogenesis in breast cancer xenografts, J. Cell Biochem, e-pub and Vogel et al. (2008) Natural and non-natural prenylated chalcones: Synthesis, cytotoxicity and anti-oxidative activity, Bioorg. Med. Chem, e-pub).
Apoptosis is an ambivalent phenomenon and in healthy tissue is considered to be damaging. It is, however, desirable for the removal of damaged cells and cancer cells. Hence apoptosis-inducing substances can be both cancer-preventive and therapeutic. The induction of apoptosis by xanthohumol could be detected in cell cultures (Gerhauser and Frank (2005), Mol. Nutr. Food Res., 49, 821823; Lust et al. (2005), Mol. Nutr. Food Res., 49, 844850; Pan et al. (2005), Mol. Nutr. Food Res., 49, 837-843; Vanhoecke (2005), Int. J Cancer, 117, 889-895; Colgate et al. (2007), Cancer Lett., 246, 201-209; Dell'Eva et al. (2007), Cancer, 110, 2007-2011; Yang et al. (2007), Apoptosis., 12, 1953-1963) and in vivo in mice (Monteiro et al. (2008) Xanthohumol inhibits inflammatory factor production and angiogenesis in breast cancer xenografts, J. Cell Biochem., e-pub) after 4-6 mg/kg per day for 60 days in the drinking water.
To grow, a tumor needs to be supplied with nutrients. Once it has reached a certain size, new blood vessels are formed for this purpose, without which the tumor's growth would stagnate. Hence angiogenesis inhibitors act to prevent cancer and are effective in cancer therapy. The anti-angiogenetic action of xanthohumol is described in cell culture (Gerhauser and Frank (2005), Mol. Nutr. Food Res., 49, 821823; Albini et al. (2006), FASEB J., 20, 527-529; Dell'Eva et al., Cancer, 110, 2007-2011) and in vivo in mice (Gerhauser and Frank (2005), Mol. Nutr. Food Res., 49, 821823; Albini et al. (2006), FASEB J., 20, 527-529; Monteiro et al. (2008), Xanthohumol inhibits inflammatory factor production and angiogenesis in breast cancer xenografts, J. Cell Biochem., e-pub) after doses of approx. 1 mg/kg per day (Albini et al. (2006), FASEB J., 20, 527-529) for three days per os, 4-6 mg/kg per day for 60 days in drinking water (Monteiro et al. (2008), J. Cell Biochem., e-pub) or 1000 mg/kg subcutaneously (Gerhauser and Frank (2005), Mol. Nutr. Food Res., 49, 821823).
In further studies on molecular mechanisms in the protective action of xanthohumol, the influence on the NF-κB (nuclear factor kappa B) was noted. NF-κB is a cellular transcription factor that plays a part in the stimulation and production of pro-inflammatory target genes such as interleukin, tumor necrosis factor α, inducible nitric oxide synthase (iNOS), and inducible cyclooxigenase (Cox-2) and has a determinant influence on progression and duration of an inflammation. The diseases featuring NF-κB-regulated genes include inflammatory events with glomerulonephritis, arteriosclerosis, septic shock or pulmonary fibrosis, as well as chronic diseases such as asthma and rheumatoid arthritis. In Albini et al. (2006), FASEB J., 20, 527-529; Colgate (2007), Cancer Lett., 246, 201-209; Dell'Eva et al. (2007), Cancer, 110, 2007-2011 and Monteiro et al. (2008), J. Cell Biochem., e-pub, the inhibition of the NF-κB is described in cell culture and in vivo.
In animal studies, xanthohumol was investigated for its action in preventing cancer. It could be demonstrated that in three different tumor models, xanthohumol inhibited formation of the tumor. Human breast tumor cells (MX-1 cells in the study Gerhauser and Frank (2005), Mol. Nutr. Food Res., 49, 821823; MXF7 cells in the study Monteiro et al. (2008), Xanthohumol inhibits inflammatory factor production and angiogenesis in breast cancer xenografts, J. Cell Biochem., e-pub) were transferred to immune-deficient mice and the growth of the tumor measured. As described in Gerhauser and Frank (2005), Mol. Nutr. Food Res., 49, 821823, following a daily subcutaneous administration of 1000 mg/kg xanthohumol, the growth of the tumor was inhibited by 46% in one week and by 83% in two weeks. As described in Monteiro et al. (2008), Xanthohumol inhibits inflammatory factor production and angiogenesis in breast cancer xenografts, J. Cell Biochem., e-pub, with a daily dose of 4-6 mg/kg xanthohumol in the drinking water, the growth of the tumor was inhibited by 35%, yet this result could not be validated statistically at the 95% level. As described in Albini et al. (2006), FASEB J., 20, 527-529, the cells of a human Kaposi's sarcoma (KS-IMM) were transferred to immune-deficient mice and treated with approx. 1.2 mg/kg xanthohumol for 23 days; the growth of the tumor was inhibited by 68%.
The anti-mutagenic/anti-genotoxic studies by Miranda et al. (2000), Cancer Lett., 149, 21-29; Dietz et al. (2005), Chem Res. Taxieal., 18, 1296-1305; Plazar et al. (2007), Mutat Res., 632, 1-8; Kac et al. (2008), Phytomedicine., 15, 216-220 and Plazar et al. (2008), Toxicol In Vitro, 22, 318-327, describe anti-mutagenic and anti-genotoxic activities of xanthohumol. Cells in culture or rat liver are pre-incubated with xanthohumol and subsequently treated with genotoxic or cancer-generating substances that form radicals. Both, in the so-called Ames test, a test for mutagenic damage in bacterial culture, and in the Comet assay, a test that describes DNA damage caused by genotoxic substances, xanthohumol could inhibit damages up to 100%, depending on doses ranging between 0.01 to 10 μM.
Xanthohumol acts against microorganisms (Herath (2003), Chem Pharm. Bull. (Tokyo), 51, 1237-1240; Buckwold et al. (2004), Antiviral Res., 61, 57-62; Wang (2004), Antiviral Res., 64, 189-194; Frolich (2005), J. Antimicrob. Chemother., 55, 883-887; Allen (2007), Avian Dis., 51, 21-26). The studies by Miranda et al. (2000), Drug Metab Dispos., 28, 1297-1302 and Frolich et al. (2005), J. Antimicrob. Chemother., 55, 883-887 describe an effect against 4 different strains of plasmodiums generating malaria and the study by Allen (2007), Avian Dis., 51, 21-26, gives a description of action against coccidian, a parasite that mainly colonises in the gastric-intestinal act of domestic animals. Xanthohumol acts in vitro as anti-viral against the HIV-1 (human immunodeficiency virus) (Wang et al. (2004), Antiviral Res., 64, 189-194), against BVDV (bovine viral diarrhoea virus), HSV-1 and 2 (herpes simplex virus) and against CMV (cytamegalovirus) (Buckwold et al. (2004), Antiviral Res., 61, 57-62).
The stability of the bones depends on equilibrium between bone-forming cells (osteoblasts) and bone-resorbing cells (osteoclasts). If the activity of the osteoclasts is proportionally greater, osteoporosis occurs. It was possible show in vitro that xanthohumol inhibits the resorption of bone by 35% in a concentration of 1 μM and by 94% with 10 μM (Tobe et al. (1997), Biosci. Biotechnol. Biochem., 61, 158-159). Another study (Effenberger et al. (2005), J. Steroid Biochem. Mol. Biol., 96, 387-399) describes the activation of osteoclasts.
Diacylglycerol acetyltransferase (DGAT) denotes an enzyme that is important for the formation and accumulation of triglycerides in the cell in the context of metabolic syndrome. The studies by Tabata et al. (1997), Phytochemistry, 46, 683-687 and Casaschi et al. (2004), J Nutr., 134, 13401346, show an inhibition of this enzyme by xanthohumol in vitro; the study reported by Nozawa et al. (2005) Biochem. Biophys. Res. Commun., 336, 754-761 was able to detect a reduction of triglycerides and of the plasma glucose level in mice.
Xanthohumol is poorly absorbed following oral application in the rat and remains largely unchanged in the excrement (Avula et al. (2004), J Chromatogr. Sci., 42, 378-382; Stevens (2004), Phytochemistry, 65, 1317-1330; Hanske (2005), Mol. Nutr. Food Res., 49, 868-873); in vitro it binds to cytosolic proteins (Pang et al. (2007), Mol. Nutr. Food Res., 51, 872-879) resulting in glucuronidation in vitro (Yilmazer (2001), FEBS Lett., 491, 252-256; Ruefer et al. (2005), Mol. Nutr. Food Res., 49, 851-856; Kim et al. (2006), J Nat Prod., 69, 1522-1524) and in vivo (Stevens (2004), Phytochemistry, 65, 1317-1330). The excrement featured a wide diversity of metabolites (Nookandeh et al. (2004), Phytochemistry, 65, 561-570), which, however, can also be derived from microbial activity in the intestine. The intestinal flora itself is not influenced in rats (Hanske et al. (2005), Mol. Nutr. Food Res., 49, 868-873).
The oral application of xanthohumol in drinking water (5×10−4M ad libitum corresponding approximately to 28 mg/kg/day) for 4 weeks showed no unfavorable impact on the health of female C3H-mice. The development of weight and the appearance of the organs, the haematologically clinical parameters, the liver enzyme values and the parameter of glucose metabolism all remained uninfluenced by xanthohumol (Vanhoecke et al. (2005), In Vivo, 19, 103-107). In a study on rats, 100 mg/kg xanthohumol was administered daily for 4 weeks in drinking water, 500 mg/kg mixed into feed and 1000 mg/kg applied over pharyngeal tube. Except for a reduction in the weight of the liver due to the breakdown of glycogen in the high-dose groups (500 mg/kg/day and 1000 mg/kg/day) no toxic effects whatsoever could be determined (Hussong et al. (2005), Mol. Nutr. Food Res., 49, 861-867). Since xanthohumol features anti-estrogen activity, investigation focused on whether reproduction with rats is impaired. The animals were treated for four weeks with 100 mg/kg/day xanthohumol in the drinking water and subsequently mated. Neither the pre-treatment of the female animals nor of the males had any influence on fertility or lactation (Hussong et al. (2005), Mol. Nutr. Food Res., 49, 861-867).
It is remarkable how extremely multi-faceted the spectrum of activity of xanthohumol is. For example, it inhibits the mechanisms of carcinogenesis in the initiation phase and in the promotion and progression phases. Anti-oxidative and anti-inflammatory characteristics are just as important as the anti-estrogen activity, the inhibition of cellular proliferation, induction of apoptosis and the inhibition of angiogenesis. All these characteristics initially found in vitro, have now also been detected in vivo in animal experiments.
The studies on acute and subchronic toxicity (4 weeks) with up to 100 mg/kg/day per os gave no indication of toxicity in mice and rats. Glycogenolysis in the rat liver after daily doses in excess of 500 mg/kg xanthohumol can easily be explained by the significantly increased energy requirement culminating in the metabolism of large quantities of xenobiotics. It should be emphasized that such high concentrations are only administered to test possible toxicity. Recent studies presented cancer-preventing effects with doses of one-digit mg/kg.
Because the mechanisms of carcinogenesis in rodents and humans are comparable, since the metabolic effects of both are very similar and many studies are conducted with recombinant human enzymes, human cells in culture or with human cells in immune-deficient mice, similar results may also be anticipated for humans.
The invention described herein is based on the primary objective to provide means by which one, several or all of the health-promoting effects of xanthohumol on humans and/or animals can be enhanced.
Xanthohumol (hereinafter also referred to as XN), a prenylflavonoid (polyphenol) of hop, occurs in the lupulin glands of the hop cones. The content of XN in hops varies depending on the hop variety between 0.1 and 1%. The hop varieties having a high content of XN usually comprise a high portion of bitter substances.
Hop products may be divided in raw hops, hop pellets and hop extracts. Since XN occurs in the lupulin glands of the hop cone, hop pellets usually exhibit a high content of XN corresponding to the accumulation of alpha-acids. Hop extracts may be obtained by extraction with CO2 and/or ethanol. Conventionally, XN enriched products are produced by a combination of both extraction methods. Depending on the production method XN contents of between 8 and 99% are achieved. The production of hop extracts enriched with XN and drinks comprising XN is described e.g. in the patents DE 19939350, DE 10256031, DE 10240065 and EP 1431385.
In the brewing process XN is relatively instable and is mainly precipitated via the trub, the yeast, by filtration and stabilization due to its limited solubility. Besides, XN is isomerized to iso-XN which also has a positive effect, however, to quite a lower extent compared to XN. With conventional methods less then 0.2 mg/l XN are reached in the final beer in most of the cases. In some stout or porter type dark beers XN contents of up to 1.2 mg/l XN were found (Walker et al., Brauwelt 2003). With a special brewing method which applies late hopping and a fast cooling of the beer wort it is possible to increase the XN content in non-filtered pale beers (DE 102 56 166).
Earlier in-house studies on behalf of the applicant, which focused on a technical field other than the subject-matter described herein, have shown that soluble roasted substances seem to be able to adsorb or bind XN and therefore apparently keep it in solution, which results in much higher yields of XN in hop extracts. In this context, a method has been provided for producing a roasted extract comprising XN from roasted products of cereals, cereal malt, coffee or cacao and a hop extract comprising XN. The hop extract comprising XN is characterized by a particularly high content of XN. Due to the use of this roasted extract, beers with a significantly increased content of XN compared to the prior art can be obtained in accordance with the German purity law, for instance (cf. EP 1 761 245 B1). Furthermore, the isomerisation of XN to iso-XN is suppressed to a large extent due to the improvement of the brewing method. Therefore, it is possible to increase dosage amounts of XN without allowing the beers to become unpleasantly bitter, for instance. Further, the addition of stabilizers is possible which would normally lead to a relevant decrease in the XN content.
While a high content of XN is also desirable in order to exploit the health-promoting effects of XN, it was first expected that the binding and/or adsorption of XN to the roasted substances would be detrimental to the beneficial activity of XN.
However, further subsequent studies directly comparing the activity of a XN containing roasted extract to the activity of pure XN now surprisingly showed that a composition containing a roasted extract and XN is even more effective than pure XN. Therefore, the interactions of the roasted substances with XN seem to not only facilitate a higher yield of XN in the extract, but furthermore they seem to enhance the beneficial activities of XN.